Melittin‐Carrying Nanoparticle Suppress T Cell‐Driven Immunity in a Murine Allergic Dermatitis Model

Abstract Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are the most common human skin disorders. Although corticosteroids have been widely used to treat ACD and AD, the side effects of corticosteroids encourage researchers to explore new immunoregulatory treatments. Here, an immunomodulatory approach based on lipid nanoparticles carrying α‐helical configurational melittin (α‐melittin‐NP) is developed to overcome T cell‐mediated inflammatory reactions in an oxazolone (OXA)‐induced contact hypersensitivity mouse model and OXA‐induced AD‐like mouse model. Intradermal injection of low‐dose α‐melittin‐NPs prevents the skin damage caused by melittin administration alone and efficiently targeted lymph nodes. Importantly, melittin and α‐melittin‐NPs restrain RelB activity in dendritic cells (DCs) and further suppresses dendritic cell activation and maturation in lymph nodes. Furthermore, low‐dose α‐melittin‐NPs leads to relief of antigen recognition‐induced effector T cell arrest in the dermis and inhibited allergen‐specific T cell proliferation and activation. Significantly, this approach successfully controls Th1‐type cytokine release in the ACD model and restricts Th2‐type cytokine and IgE release in the AD‐like model. Overall, intradermal delivery of low‐dose α‐melittin‐NPs efficiently elicits immunosuppression against T cell‐mediated immune reactions, providing a promising therapeutic strategy for treating skin disorders not restricted to the lesion region.

(concentration of α-peptide, 0.429 mg kg -1 ) in PBS were intradermally injected in the ACD model. PBS was intradermally injected as control.
Culture of BMDCs and BMDMs: C57BL/6 mice were sacrificed, and the femurs and tibias were dissected using scissors. The bones were flushed with a syringe filled with RPMI-1640 medium to isolate Bone Marrow (BM) cells. BM cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U mL -1 penicillin-streptomycin, 50 μmol L -1 βmercaptoethanol, 20 ng mL -1 murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 ng mL -1 IL-4 (PeproTech). On day 3 and day 6, half of the supernatant was gently removed and replaced with the same volume of the medium. BMDCs were obtained by collecting nonadherent cells and purified with 30% Percoll (Sigma-Aldrich) to achieve the best viability on day 9. BMDCs were further verified by evaluating the expression of CD11c using flow cytometry. Attached cells were collected by washing with ice-cold PBS and incubating them for 30 min on ice to obtain BMDMs. BMDMs were further verified by confirming high expression of CD11b and F4/80 using flow cytometry. BMDCs or BMDMs were then seeded in 96-well plates, stimulated with DNBS (10 μg mL -1 ) or LPS (100 ng mL -1 ) and cocultured with α-melittin-NPs, α-peptide-NPs or melittin for 24 h. The BMDCs or BMDMs were then harvested, and the expression of the costimulatory molecules CD80 and CD86 was evaluated using flow cytometry. BMDCs were incubated with LPS and each reagent at various doses (1 μmol L -1 , 2.5 μmol L -1 , or 5 μmol L -1 ) for 60 min to measure pRelA and pRelB levels. The BMDCs were then pre-stained with anti-mouse antibodies specific for CD11c (BV421, clone N418) and MHCII (Percp5.5, clone M5/114.15.2). The pre-stained BMDCs were then fixed at 4 °C for 20 min (avoiding lights) and subsequently stained with anti-mouse pRelA (Ser536, 93H1) and pRelB (Ser552, D41B9) antibodies according to the manufacturer's instructions (CST).

Investigation of the toxicity of melittin and α-melittin-NPs in vivo:
Ten microliters of 0.14, 0.70, or 3.52 nmol melittin or α-melittin-NPs were intradermally injected into one ear, and PBS was injected into the contralateral ear. Ear thickness was measured with a dial micrometer at 12 h after injection. The increase in ear thickness was calculated as follows: (ear thickness after melittin or α-melittin-NP injection -ear thickness before melittin or αmelittin-NP injection) -(ear thickness after PBS injection -ear thickness before PBS injection).

Measurements of degranulation:
Degranulation was monitored by measuring the release of histamine in the supernatants. P815 cells were collected and washed twice with PBS by centrifugation at 300 × g for 5 min at 4 °C and resuspended in a high glucose DMEM medium containing 10% heat-inactivated FBS. The cells were then treated with various concentrations (0.01-5 μmol L -1 ) of α-peptide-NPs, α-melittin-NPs or melittin and incubated for 30 min at 37 °C. The supernatants were then collected to detect the amount of histamine released. An ELISA was performed according to the manufacturer's manual. The O.D. of the samples was detected with a UV-visible absorbance microplate reader at a wavelength of 450 nm.
Whole-field fluorescence imaging and intravital imaging: Mice were anesthetized by administering an intraperitoneal injection of 0.2 mg ketamine per gram of body weight and 0.02 mg xylazine per gram of body weight. An in-house whole-field fluorescence imaging system [3] was used to image the α-melittin-NP distribution. A BP 716/40 excitation filter and BP 800/40 emission filter were used for imaging α-melittin-NPs (DiR-BOA). For the intravital imaging of T cells and DCs in dermis. T cells were isolated from OXA-sensitized mice (MACS, Stemcell Tech) and labeled with the cell tracker dye CMTMR (Invitrogen) and intravenously injected into CD11c-Venus mice. All imaging was performed in a field of 424 μm × 424 μm (512 × 512 pixels) with excitation wavelengths of 488 nm for Venus and 561 nm for CMTMR. Images were acquired at a rate of two frames per minute over a z-range of 50 μm (below the epidermis) by performing 10-µm z-steps. More details about the intravital imaging protocol, three-dimensional reconstruction and imaging data analysis refer to our previous work [4] .

Distribution of α-melittin-NPs in organs and tissues after injection:
FITC-labeled αmelittin-NPs or PBS were intradermally injected into the tail base region of mice in the ACD model. The skin at the injection site and other organs were collected and weighed from the ACD mice at 24 h, 48 h, and 72 h. Next, the tissues were mechanically digested in 1 mL PBS by surgical shears and tissue homogenizer to fully release the FITC signal. The organ tissue suspension after grinding was then centrifuged at 3,000 rpm for 5 min, and 500 μL supernatant was collected for following FITC measurement. Due to the tissue suspension of the liver and brain was extremely thick, the supernatant from the liver and brain was diluted 10 times before the FITC measurement. After that, the supernatant was further centrifugation at 12,000 rpm for 10 min, and 100 μL supernatant was placed in a 96-well plate to detect the FITC emission using a FlexStation 3 multi-mode microplate reader (Molecular Devices, CA, USA).
Cytokine and chemokine quantitation: Total cytokine levels in mouse serum or cell culture supernatant were analyzed using specific ELISA kits (IFN-γ: BioLegend). IgE levels in mouse serum were analyzed with a specific ELISA kit (BioLegend). The local levels of TNF-α, IL-1β, IL-4 and IFN-γ in skin lysates were analyzed with specific ELISA kits (BioLegend). Briefly, an ELISA plate was coated with anti-mouse IFN-γ, IL-4, TNF-α, IL-1β, or IgE monoclonal antibodies (mAbs). The ELISA plates were then washed three times with Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). Diluted serum samples were added to the wells and incubated for 2 h. After washes with TBS-T, biotinylated antimouse IgE was added to each well and incubated for 1 h. After washes with TBS-T, the wells were incubated with avidin-conjugated HRP (BD Bioscience) for 30 min and then washed with TBS-T. The reaction was developed with a substrate chromogen for 30 min and stopped by adding an equal volume of 2 mol L -1 H 2 SO 4 . The optical absorbance at a wavelength of 450 nm was measured. The reaction product of mouse IFN-γ/IgE was used as the standard. The skin was harvested and weighed to measure tissue cytokines in the AD model. Then, tissue samples were minced and lysed in lysis buffer containing PBS and 1% Triton X freshly supplemented with a protease inhibitor cocktail (Sigma-Aldrich). The lysates were then filtered, aliquoted, and stored at −80 °C until analysis. Samples were assayed using a LEGENDplex TM Mouse Th Cytokine Panel (12 plex, BioLegend) according to the manufacturer's instructions. The data were analyzed using LEGENDplex software (BioLegend).

Quantitative PCR analysis:
Total RNA of spleen or ear was extracted using TRIZOL reagent (Invitrogen) and the extraction procedure followed the instructions provided by the manufacturer. The concentration and quality of RNA were quantified by using Nanodrop (Invitrogen). RNA was reverse transcribed into cDNA using the PrimeScript RT Mast Mix kit (TaKaRa) by referring to the protocol provided by the manufacturer. Real-time quantification of PCR was performed by using TB Green Premix Ex TaqTM  Biochemical analyses: Blood samples were collected before the mice were sacrificed and then incubated at 4 °C overnight and centrifuged to obtain mouse serum. Serum biochemical analyses were performed using a biochemical analyzer (SPOTCHEM EZ SP-4430, Arkray Inc., Kyoto, Japan).

Histopathological analyses of skin, liver and kidney in AD models:
The skin, livers, and kidneys were extracted from normal or treated mice and fixed in a 4% paraformaldehyde solution. The organs were embedded in paraffin, sectioned, and stained with H&E. The H&Estained slices were scanned with a Nikon Ni-E (Nikon, Minato, Tokyo, Japan), and images were acquired using NIS-Elements software and further analyzed with ImageJ software.   Quantification of the IL-1β concentration in each group. The ears from the ACD mice in the PBS-, α-melittin-NP-, DEX-, melittin-and α-peptide-NP-treated groups were harvested (n = 4 mice per group). Total protein was extracted to assess local cytokine levels in inflamed ear tissue. ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001; one-way ANOVA followed by Bonferroni's post hoc test. Figure S4. α-Melittin-NPs are mainly restricted to the injected skin and skin-draining LNs (ILN, ALN, DLN). Mice were sensitized with 2% (W/V) OXA in day 0, and challenged with 0.3% (W/V) in day 5. 20 nmol of FITC-α-melittin-NPs (quantification was based on FITC content) were intradermally injected at the tail base 30 min before the rechallenge of OXA. Skins and other organs were collected, weighed, and mechanically digested in PBS for 3 min with an electronic homogenizer. The tissues were centrifuged at 12,000 g for 5 min, and the supernatants were collected to detect FITC content using a FlexStation 3 multi-mode microplate reader (Molecular Devices, CA, USA). N = 3 per group, means ± SEM.           The spleens from AD model mice were collected, and mRNA was extracted to measure the expression of IgE and IgM in PBS, DEX, melittin, α-melittin-NP, and α-peptide-NP groups. A) Spleen index in 5 groups, in which spleen weights were divided by body weights to get the spleen index. B) IgE relative expression in PBS, DEX, melittin, α-melittin-NP, and αpeptide-NP groups (n ≥ 3 samples per group, means ± SEM). C) IgM relative expression in PBS, DEX, melittin, α-melittin-NP, and α-peptide-NP groups. * p < 0.05, one-way ANOVA followed by Bonferroni's post hoc test in (B, C).

Supporting Figures and Figure Legends
Figure S16. T cell and B cell percentage in different immune organs in AD mice with α-Melittin-NPs treatment. T cell and B cell percentage in different immune organs in AD mice with α-Melittin-NPs treatment (n ≥ 3 samples per group, means ± SEM). The mice of AD models with different treatments were sacrificed and organs were harvested to analyze the T cells and B cells populations. A-C) B cells (A), CD4 + T cells (B), and CD8 + T cells (C) in the spleen, dLN, blood, and BM were analyzed by flow cytometry. D) The histograms display the CD4/CD8 T cells ratio in the spleen, dLN, blood, and BM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; one-way ANOVA followed by Bonferroni's post hoc test in (A-D).